Elsevier

Metabolic Engineering

Volume 75, January 2023, Pages 78-90
Metabolic Engineering

Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

https://doi.org/10.1016/j.ymben.2022.10.016Get rights and content

Highlights

  • Growth of C. necator H16 on formate was improved via adaptive laboratory evolution, genome sequencing, and engineering.

  • Deletion of the regulator phcA and the megaplasmid pHG1 led to increased growth rates on formate and other carbon sources.

  • RNA-seq transcriptomics of the PhcA quorum sensing system revealed regulation of motility, adhesion, and protein secretion.

  • A genome-streamlined C. necator strain will serve as a chassis for biological upgrading of formate derived from CO2.

Abstract

Conversion of CO2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO2, has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin–Benson–Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. The metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ΔphcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

Introduction

Atmospheric concentrations of carbon dioxide have reached the highest levels present on Earth for several million years and are steadily increasing. In order to avert the catastrophic effects of climate change, global civilization must rapidly deploy technologies capable of reducing emissions of CO2 and other greenhouse gases toward net zero levels (Masson-Delmotte et al., 2021). One strategy entails capturing and converting CO2 at the point of emission, such as a variety of industrial waste gas streams, where CO2 is available at a relatively high concentration. Using renewable sources of electricity, electrolysis systems have the potential to electrochemically reduce CO2 to a multitude of products including carbon monoxide, formate, ethanol, ethylene, and other hydrocarbons (Jhong et al., 2013).

In particular, highly efficient electrochemical reduction of CO2 to formate and formic acid has been demonstrated by multiple groups (Chen et al., 2020; Fan et al., 2020; Yang et al., 2020; Zheng et al., 2021). Formic acid is itself a valuable commodity used in various agricultural, chemical, pharmaceutical, and textile industries. Recently, formate has also gathered significant interest as a potential feedstock for microbial upgrading, as it can be consumed as the sole source of carbon and energy by some microbial species, termed formatotrophs (Cotton et al., 2020; Satanowski and Bar-Even, 2020; Yishai et al., 2016). It is also highly water soluble, which enables microbial conversion without the safety, transport, solubility, and mass-transfer challenges associated with gaseous feedstocks. Therefore, it is an ideal intermediate molecule to serve as a bridge between biological and electrochemical conversion technologies. Within the envisioned “formate bioeconomy,” cheap renewable electricity produced at off-peak hours could be used to convert CO2 to formate, which can be stored, and later converted by metabolically engineered microbes into a virtually limitless spectrum of fuels, chemicals, and materials (Yishai et al., 2016).

Cupriavidus necator (formerly known as Ralstonia eutropha, Alcaligenes eutrophus, Wautersia eutropha, and Hydrogenomonas eutropha) is one of the best-studied native formatotrophs. C. necator is able to grow autotrophically using the Calvin-Benson-Bassham (CBB) cycle to fix CO2 from its environment when an energy source such as H2 is also provided (Schwartz et al., 2003). C. necator is also capable of growth on formate as its sole source of carbon and energy, where intracellular formate dehydrogenation is carried out by several native formate dehydrogenases to generate both energy in the form of NADH reducing equivalents and CO2 for assimilation by the CBB cycle (Cramm, 2009; Pohlmann et al., 2006). C. necator is amenable to formate concentrations up to at least 2 g/L, and the effects of formate toxicity can be mitigated in pH-controlled fed-batch cultivations (pH-stat) that maintain a low concentration of formic acid (Grunwald et al., 2015). C. necator is also genetically tractable (Bi et al., 2013; Tee et al., 2017; Xiong et al., 2018), has been successfully engineered to produce myriad products (Lu et al., 2016; Raberg et al., 2018), and has long been employed in large-scale and high cell density commercial production of polyhydroxyalkanoate (PHA) biopolymers (Byrom, 1987, 1992; Kourmentza et al., 2017). Recently, this species has been metabolically engineered to autotrophically produce a variety of chemicals from CO2 (Panich et al., 2021) including: methyl ketones (Müller et al., 2013), alka(e)nes (Crépin et al., 2016), terpenes (Krieg et al., 2018), acetoin (Windhorst and Gescher, 2019), fatty acids (Li et al., 2019), isopropanol (Garrigues et al., 2020), lipochitooligosaccharides (Nangle et al., 2020), sucrose (Nangle et al., 2020), polyhydroxyalkanoates (Nangle et al., 2020; Tanaka et al., 2021), 1,3-butanediol (Gascoyne et al., 2021), trehalose (Löwe et al., 2021), D-mannitol (Hanko et al., 2022), glucose (Wang et al., 2022), and lycopene (Wu et al., 2022), as well as isobutanol and 3-methyl-1-butanol from electrochemically generated formate (Li et al., 2012). Additionally, progress has been made towards improving autotrophic growth of C. necator via optimization of its native metabolism, and by introduction of heterologous enzymes or pathways (Claassens et al., 2020; Dronsella et al., 2022; Li et al., 2020).

As a soil bacterium, C. necator evolved in an environment with variable and transitory sources of carbon and energy. Consequently, it has been suggested that its genome is that of a strong generalist, with a diverse chemolithotrophic metabolism capable of versatile growth on a wide variety of substrates and electron acceptors (Pohlmann et al., 2006; Schwartz et al., 2003; Volodina et al., 2016). As such, we hypothesized that wild-type C. necator H16 is unlikely to be fully optimized for growth on formate as the sole source of carbon and energy. Indeed, recent analysis of protein allocation and utilization during growth on several substrates, including formate, suggested that large fractions of the proteome are underutilized, and that autotrophy may be a recent evolutionary acquisition in H16 (Jahn et al., 2021).

The genetic, physiologic, and molecular mechanisms underlying formatotrophy are not fully understood, making rational metabolic engineering to improve conversion of formate difficult. In situations such as this, adaptive laboratory evolution (ALE) is a powerful tool for generating desirable phenotypic improvements without complete, a priori knowledge of the mechanisms that govern them (Portnoy et al., 2011; Winkler et al., 2013). The emergence of low-cost full-genome sequencing and bioinformatics have enabled relatively facile identification and evaluation of potentially causative mutations and have elevated ALE as a tool for biological discovery and strain development (Sandberg et al., 2019).

In this work, we aimed to improve C. necator H16 as a host for formate conversion. To this end, we first subjected it to ALE using serial batch transfers with formate as the sole source of carbon and energy, in order to naturally select for mutations that enabled cells to grow more rapidly. Evolved isolates were analyzed by whole genome sequencing to identify genetic targets for rational metabolic engineering. We then generated a series of rationally engineered strains (Table 1) and found that they recapitulated and ultimately exceeded the growth improvements observed in the evolved strains. RNA-seq transcriptomics were performed on engineered strains to help elucidate the underlying mechanisms that contributed to improved growth on formate. We found deletion of the gene encoding the transcriptional regulator PhcA, the soluble and membrane-bound hydrogenase operons, the megaplasmid copy of the CBB operon, and finally the entire megaplasmid pHG1, were the most effective genetic modifications. Collectively, these results point towards genome minimization as a promising strategy for generating C. necator strains with improved growth under controlled conditions. Surprisingly, we also found that modifications that improved growth on formate also improved growth on succinate and fructose, yielding an improved C. necator platform strain with substantial academic and industrial potential.

Section snippets

Plasmid construction

Plasmid synthesis using the pK18sB vector (GenBank Accession MH166772, Addgene Plasmid # 177838) backbone was performed by Twist Biosciences. Conjugative plasmids were built using the compact conjugation vector pK18msB (GenBank Accession # OK423783, Addgene Plasmid #177839) (Ling et al., 2022). For plasmids built manually, Phusion Polymerase (New England Biolabs) was used for amplifying fragments from C. necator genomic DNA. Plasmids were assembled via the Gibson Method using Gibson Assembly

Adaptive laboratory evolution and whole genome sequencing reveal targets for improving formatotrophy

Cupriavidus necator is a metabolic generalist, capable of adapting to variable resources and dynamic conditions and, consequently, it is likely not optimized for growth on formate alone. Therefore, we hypothesized that its growth on formate could be improved upon using ALE.

In order to select for random genetic mutations that improve growth on formate, we performed ALE of C. necator in six separate lineages grown in parallel on minimal medium containing 50 mM sodium formate as the source of

Deletion of the megaplasmid copy of the CBB operon

Whole genome sequencing of the ALE strains occasionally produced surprising results. For example, we found partial or total loss of the pHG1 copy of the CBB operon in 5 out of 6 sequenced isolates. Assuming that mutations that are most useful for improving formate utilization are more likely to appear in multiple lineages, these results suggest that there was a strong evolutionary incentive to lose the CBBp copy of the operon. This is a very surprising result, considering that the CBB cycle is

Conclusion

Inspired by the results of adaptive laboratory evolution, and using the power of rational metabolic engineering, we were able to develop a new platform strain of C. necator, CHC124 (ΔpHG1 ΔphcA ΔphaCAB), with improved growth characteristics. Deletion of the megaplasmid pHG1 (6.1% of the genome) and the quorum-sensing transcriptional regulator PhcA enabled maximum growth rates on formate that, to our knowledge, exceed any previously published results. These modifications also increased growth

Author statement

Christopher H. Calvey: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization, Violeta Sànchez i Noguéa: Conceptualization, Methodology, Formal analysis, Writing - Review & Editing, Visualization, Supervision, Aleena M. White: Investigation, Formal analysis, Writing - Review & Editing, Colin M. Kneucker: Investigation, Sean P. Woodworth: Investigation, Hannah M. Alt: Investigation, Carrie A. Eckert: Conceptualization,

Declaration of competing interest

C.H.C. and C.W.J have submitted a patent application on engineered strains related to this work.

Acknowledgements:

This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding was provided by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting

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