Biological production of chemicals from alternative carbon feedstocks, such as carbon dioxide (CO₂), is pivotal to the development of sustainable chemical processes. Today, a majority of biologically produced chemicals are obtained via engineered heterotrophs, such as the workhorse organism Escherichia coli that converts biomass feedstock to fuels and chemicals.(1) An implicit assumption in this process is that plants will harvest energy from sunlight and fix CO₂ from the environment to generate the biomass that E. coli will use for chemical production. Therefore, biological production of chemicals from renewable biomass is dependent on the price of plant biomass (e.g., corn bushel price), the organism’s growth rate, and its productivity. Engineering photoautotrophs, such as cyanobacteria and microalgae, which fix CO₂ using sunlight, permits circumvention of the reliance on plant feedstock and has been used to produce biofuels. However, E. coli currently has several advantages over photoautotrophs, including a growth rate that is 5 times faster than that of cyanobacteria, a more sophisticated synthetic biology toolbox for engineering metabolic pathways, and independence from light, which not only reduces the cost of the bioreactor but also increases the productivity of the system. One of the Grand Challenges in metabolic engineering has been to port the CO₂ fixing capabilities of autotrophs to a workhorse heterotroph, such as E. coli. An autotrophic E. coli could leverage the knowledge of more than 100 metabolic pathways already optimized for the production of fuels and chemicals and produce them from CO₂. Milo group’s 2016 attempt to port CO₂ fixation into E. coli achieved a 35% CO₂ fixation into biomass.(2) Three years later, in their 2019 contribution, Gleizer et al. engineered an E. coli capable of using CO₂ as its sole carbon source.(3) The authors harnessed metabolic engineering to build an E. coli with disrupted glycolysis and pentose phosphate pathways, capable of fixing CO₂ via the Calvin cycle, and using formate as the electron source. This strain was then optimized for CO₂ fixation via adaptive evolution, over 350 days, resulting in mutations that regulate gene expression and pathway regulation. The introduction of formate into the growth media, and formate dehydrogenase as a method for producing reducing power, proved to be the key additions for improving from 35% to 100% of biomass from CO₂. The adaptive evolution step was fundamental to achieve an E. coli that uses CO₂ as its sole carbon source. To rationally engineer such an E. coli would have required the design, building, and testing of an extremely large number of constructs, as this problem requires replacing a central metabolic pathway (the glycolysis/pentose phosphate pathway) with a heterologous one (the Calvin cycle). This is orders of magnitude more difficult than engineering a chemical production pathway to siphon central metabolism intermediates (e.g., pyruvate) for chemical production. In the latter, one need only up- or downregulate the central metabolism, not replace it. More and more, we are seeing adaptive (or directed) evolution being applied to metabolic engineering challenges, and Gleizer et al. show that, as long as we can link the desired activity to biomass accumulation, we can replace the central metabolism in biological systems. An exciting application of this work is the availability of a new, more efficient, autotrophic host for chemical production. The increased efficiency comes from the use of formate, as opposed to light, to generate reducing power. As a light-independent organism, the engineered E. coli is not limited by the low efficiency of light utilization(4) and is instead capable of utilizing all of the exogenously fed formate toward biomass production through carbon fixation.(3) Albeit, the current strain exhales more CO₂ than it consumes (Figure 1). Figure 1. Advancing the potential for the production of chemicals from carbon dioxide in E. coli. Several steps remain before the autotrophic E. coli can be implemented for chemical production at scale. The autotrophic E. coli doubling time will need to be shortened (18 h, as compared to 30 min for the wild type). Also, the Type II RuBisCo used by Gleizer et al. has a high k_cat yet low selectivity for CO₂ over O₂, which at the 10% CO₂ concentration used in the chemostat pushed the selectivity toward the carbon fixation reaction. In bioreactors, a high CO₂ concentration can be achieved via fed gas. However, for applications under atmospheric conditions or in cases in which CO₂ inhibits the reaction, a Type I RuBisCO, with a higher selectivity for CO₂ and intermediate k_cat values, will need to be used. Another way to improve carbon fixation in a Type II RuBisCO system is through a carbon-concentrating mechanism. In bacteria, this often comes in the form of a carboxysome or carboxysome-like bacterial microcompartment (BMCs). While they are not natively found in E. coli, recent advancements in heterologous expression and customized cargo loading of BMCs suggest that this is a viable solution for this system.(5) Increasing the local CO₂ concentration can compensate for lower selectivity while taking advantage of high turnover numbers in Type II RuBisCO. Increasing CO₂ levels in Earth’s atmosphere present the challenge of how to capture this greenhouse gas, with a goal of addressing the effects of global climate change. The ability to convert the captured carbon into a value-added product is appealing for carbon capture and sequestration as well as for sustainable chemical production and reduction of our dependence on nonrenewable fossil fuels. The successful engineering of a fully autotrophic E. coli strain opens the door for the use of a fast-growing model organism in carbon capture and production of chemicals and biofuels, offering distinct advantages over currently available microbes such as algae and cyanobacteria. Gleizer et al.’s accomplishment presents a first, but crucial step, on the path toward sustainable production of value-added chemicals from CO₂. N.S.K. is funded by a Georgia Institute of Technology Renewable Bioproducts Institute Graduate Fellowship. The authors declare no competing financial interest. This article references 5 other publications.

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推进在大肠杆菌中由二氧化碳生产化学品的潜力。

由替代碳原料（例如二氧化碳（CO ₂））进行生物化学生产对于发展可持续的化学过程至关重要。如今，大多数生物生产的化学药品都是通过工程异养生物获得的，例如将生物量原料转化为燃料和化学药品的主力生物大肠杆菌（1）。这一过程的一个隐含假设是，植物将通过日光吸收能量并固定二氧化碳。₂从环境中产生大肠杆菌的生物量将用于化工生产。因此，由可再生生物质生产化学药品的生物取决于植物生物质的价格（例如玉米蒲式耳价格），生物的生长速度及其生产率。工程光合自养生物，例如蓝细菌和微藻类，利用阳光将CO ₂固定下来，从而可以规避对植物原料的依赖，并已用于生产生物燃料。但是，大肠杆菌与光合自养生物相比，目前具有若干优势，包括比蓝细菌快5倍的生长速度，用于工程化代谢途径的更复杂的合成生物学工具箱以及不受光的影响，这不仅降低了生物反应器的成本，而且还增加了生物反应器的成本。系统的生产力。代谢工程学的重大挑战之一是将自养生物的CO ₂固定能力移植到强力异养生物（如大肠杆菌）上。自养大肠杆菌可以利用对100多种代谢途径的知识，这些途径已经针对燃料和化学物质的生产进行了优化，并通过CO _2进行生产。Milo Group在2016年尝试移植CO ₂固定到大肠杆菌实现了35％的CO ₂固定成生物质。（2）三年后，在他们的2019贡献，Gleizer等。设计了一种能够将CO ₂用作唯一碳源的大肠杆菌。（3）作者利用代谢工程技术构建了一种具有被破坏的糖酵解和戊糖磷酸途径，能够通过加尔文循环固定CO _2的大肠杆菌，并利用甲酸盐作为电子源。然后针对CO ₂优化该菌株在超过350天的时间内通过适应性进化进行固定，从而产生了可调控基因表达和途径调控的突变。甲酸被引入到生长培养基中，以及甲酸脱氢酶作为产生还原力的方法，被证明是将CO ₂的生物质从35％提高到100％的关键添加剂。适应性进化步骤对于获得使用CO ₂作为唯一碳源的大肠杆菌至关重要。合理设计这样的大肠杆菌本来需要设计，构建和测试大量构建体，因为此问题需要用异源途径（加尔文循环）代替中心代谢途径（糖酵解/戊糖磷酸途径）。这比设计一条化学生产路径来虹吸中心代谢中间体（例如丙酮酸）进行化学生产要困难几个数量级。在后者中，只需要上调或下调中央代谢，而不是替代它。越来越多地，我们看到自适应（或定向）进化被应用于代谢工程学的挑战，Gleizer等人。表明，只要我们可以将所需的活性与生物量积累联系起来，就可以替代生物系统中的中枢代谢。这项工作令人兴奋的应用是新的，更高效，自养的化学生产宿主。效率的提高是由于使用甲酸盐而不是光来产生降低的功率。作为一种与光无关的生物，大肠杆菌不受光利用效率低的限制（4），而是能够通过碳固定将所有外源甲酸酯利用来进行生物量生产。（3）尽管目前的菌株呼出的CO ₂比其消耗的多。 （图1）。图1.提高在大肠杆菌中由二氧化碳生产化学药品的潜力。在将自养大肠杆菌用于大规模化学生产之前，还需要完成几个步骤。自养大肠杆菌的倍增时间将需要缩短（18小时，而野生型为30分钟）。另外，Gleizer等人使用的II型RuBisCo。k_猫高，但对CO _2的选择性低超过O _2的浓度，在化学稳定剂中使用的O ₂浓度为10％CO ₂将选择性推向了碳固定反应。在生物反应器中，可通过进料气体实现高CO ₂浓度。然而，对于在大气条件下或在CO ₂抑制反应的情况下的应用，将需要使用对CO _2的选择性更高且具有中等k _cat值的I型RuBisCO 。在II型RuBisCO系统中改善碳固定性的另一种方法是通过碳浓缩机制。在细菌中，这通常以羧基体或类似羧基体的细菌微室（BMC）的形式出现。虽然它们不是本地存在的大肠杆菌，BMC的异源表达和定制货物装载的最新进展表明，这是该系统的可行解决方案。（5）增加局部CO ₂浓度可以补偿较低的选择性，同时利用II型中的高周转率RuBisCO。为了解决全球气候变化的影响，地球大气中CO ₂含量的增加提出了如何捕获这种温室气体的挑战。将捕获的碳转化为增值产品的能力吸引了碳的捕获和封存，以及可持续的化学生产以及减少了我们对不可再生的化石燃料的依赖。完全自养大肠杆菌的成功工程菌株为在碳捕获，化学物质和生物燃料的生产中使用快速增长的模型生物打开了大门，与目前可用的微生物（例如藻类和蓝细菌）相比，它具有明显的优势。Gleizer等人的成就代表了从CO ₂可持续生产增值化学品的道路上的第一步，但至关重要。NSK由佐治亚理工学院可再生生物产品研究所研究生奖学金资助。作者宣称没有竞争性的经济利益。本文引用了其他5个出版物。