Engineering Carboxylic Acid Reductase (CAR) via NADPH Recycling

Xuan Le Author

04/02/2021 Added

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Rapid evolution of enzyme activities is often hindered by the lack of efficient and affordable methods to identify beneficial mutants. This video discusses the development of a new growth-coupled selection method for evolving NADPH-consuming enzymes based on the recycling of this redox cofactor. The method relies on a genetically modified E. coli strain, which overaccumulates NADPH. This method was applied to the engineering of a carboxylic acid reductase (CAR) for improved catalytic activities on adipate. Mutant enzymes with up to 17-fold improvement in catalytic efficiency were identified from single-site saturated mutagenesis libraries.

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[00:00:02.070]Hello, everyone.
[00:00:02.950]Welcome to my presentation.
[00:00:04.830]My name is Xuan Le, and I am an undergraduate student
[00:00:07.950]studying chemical engineering here at UNL.
[00:00:10.770]Today, I will be talking about the engineering
[00:00:13.180]of carboxylic acid reductase, or CAR enzymes,
[00:00:16.920]via NADPH recycling.
[00:00:21.010]A large number of chemical synthetic processes
[00:00:23.990]used in our world
[00:00:25.030]produce or depends on toxic materials.
[00:00:28.410]Enzymes are biological solutions
[00:00:30.280]to these traditional chemical synthesis
[00:00:32.670]because they catalyze useful reactions
[00:00:35.550]and usually require only mild reaction conditions.
[00:00:39.910]CAR enzymes selectively reduce carboxylic acids
[00:00:43.570]into their corresponding aldehydes.
[00:00:46.540]Aldehydes are mainly used as perfumes, flavoring agents,
[00:00:50.550]and they can also be easily converted
[00:00:53.220]into other products such as alcohols and amines,
[00:00:56.840]which can be used in polymer production.
[00:01:00.050]Figure 1 shows the domain architecture
[00:01:02.300]and catalytic cycle of CAR.
[00:01:04.700]CAR has three domains: an adenylation domain shown in blue,
[00:01:09.540]a reduction domain shown in red,
[00:01:12.030]and an internal phosphopantetheine attachment domain
[00:01:15.840]shown in orange.
[00:01:17.940]CAR uses ATP and NADPH as cofactors.
[00:01:23.360]The chemical reactants of enzymes are called substrates,
[00:01:26.810]and previous works have shown that CARs
[00:01:29.470]have broad substrate specificities.
[00:01:32.651]What this means is that CAR can be used
[00:01:34.740]to synthesize a wide range of chemicals.
[00:01:37.610]However, CARs have poor kinetic properties
[00:01:40.440]on substrates of interest, mainly dicarboxylic acids
[00:01:44.200]like adipates, that are common in nature.
[00:01:48.060]In this work, an NADPH-overaccumulating host strain,
[00:01:52.140]delta-2 was designed.
[00:01:54.280]This strain was used as a selection platform
[00:01:57.070]to identify CAR mutants with improved kinetic properties
[00:02:00.590]on the desired substrate adipate.
[00:02:03.570]Some experimental procedures performed
[00:02:05.810]include single-site saturation mutagenesis,
[00:02:09.430]growth-coupled selection of CAR mutant libraries,
[00:02:12.410]protein purification, and biochemical assays.
[00:02:16.840]We started the engineering process
[00:02:18.930]by first constructing a suitable host,
[00:02:21.820]as shown in Figure 2A.
[00:02:23.860]An E. coli strain that overaccumulates NADPH
[00:02:27.520]was designed by deleting two genes,
[00:02:30.590]resulting in a new strain called delta-2.
[00:02:34.320]When cultured in minimal media containing glucose
[00:02:37.550]as the carbon source, we saw that delta-2
[00:02:40.490]had extended lag phase compared to wild-type E. coli,
[00:02:44.890]and this can be seen in Figure 2B.
[00:02:48.130]This curve labeled "wt" represents the growth curve
[00:02:52.450]of the wild type.
[00:02:53.980]And this gray curve represents that of delta-2.
[00:02:58.460]And you can see clearly that delta-2
[00:03:00.690]had a much longer lag phase comparing to the wild type.
[00:03:05.900]We hypothesize that delta-2's long lag phase
[00:03:08.910]was because of toxic overaccumulation of NADPH.
[00:03:13.340]And in order to fix this,
[00:03:15.200]we introduced the Mycobacterium avium CAR, or MavCAR,
[00:03:20.040]into delta-2.
[00:03:21.820]As a reminder, CAR uses NADPH,
[00:03:25.270]so it will consume the overaccumulated NADPH
[00:03:29.510]when expressed in delta-2 in the presence
[00:03:32.500]of a carboxylic acid substrate.
[00:03:35.920]As shown in Figure 2B, this was the case.
[00:03:40.005]MavCAR-delta-2 had a much shorter lag phase
[00:03:44.560]comparing to delta-2 in the presence benzoate,
[00:03:48.830]which is a native substrate of CAR.
[00:03:52.150]This result confirms
[00:03:53.810]that the CAR-catalyzed reaction
[00:03:56.080]recovered the growth of delta-2 cells.
[00:03:59.980]Next, we looked at the correlation
[00:04:02.070]between growth rate of delta-2
[00:04:04.330]and the activity level of CAR.
[00:04:06.920]Cells were cultured on media plates
[00:04:09.250]containing glucose as the carbon source
[00:04:11.980]and different concentrations
[00:04:13.570]of 6-hydroxyhexanoate substrate.
[00:04:16.990]Looking at Figure 3,
[00:04:18.810]number 1 represents delta-2,
[00:04:21.060]in which there is no growth.
[00:04:23.570]Number 4 represent wild type,
[00:04:25.820]which acted as a positive control.
[00:04:28.930]And most importantly, number 2 and 3
[00:04:31.520]represent growth of delta-2 strains expressing MavCAR.
[00:04:36.460]At higher concentrations, we saw faster growth.
[00:04:40.940]This positive correlation
[00:04:42.500]between CAR activities and cell growth
[00:04:44.810]demonstrated that E. coli delta-2
[00:04:47.433]is an appropriate host for detecting CAR variants
[00:04:51.170]with improved activities.
[00:04:54.520]So to determine if cell growth could be used
[00:04:57.460]to identify MavCAR mutants with improved kinetic properties,
[00:05:02.100]we generated mutants at the MavCAR active site,
[00:05:05.230]which is the region responsible
[00:05:07.080]for binding the carboxylic acid substrate.
[00:05:10.250]The 10 residues labeled in black in Figure 4 were mutated
[00:05:14.200]because we presumed that they were important
[00:05:16.690]for binding of desired substrate adipate.
[00:05:20.350]We transformed the delta-2 host
[00:05:22.320]with each of the 10 libraries.
[00:05:24.870]Then we plated the transformed cells
[00:05:26.850]on media plates containing glucose and adipate.
[00:05:30.620]We monitored colony formation within a 72-hour period,
[00:05:35.010]and only colonies with faster growth rates
[00:05:37.870]than the control were picked.
[00:05:39.750]DNA sequencing analysis were then performed
[00:05:42.680]on these chosen colonies in order to identify mutations.
[00:05:47.300]The chosen mutants were expressed, purified,
[00:05:50.000]and characterized by kinetic assays.
[00:05:53.040]Table 1 shows kinetic activities
[00:05:55.330]of the chosen mutants on adipate.
[00:05:57.970]Catalytic efficiency kcat/ Km
[00:06:01.620]is the best value to represent the enzyme's overall ability
[00:06:05.510]to convert substrate to product.
[00:06:08.170]Most of the identified mutants had improved efficiency
[00:06:12.260]with the best mutant being N335R,
[00:06:16.370]as it had a 17-fold improvement in catalytic efficiency
[00:06:21.790]comparing to that of the wild type.
[00:06:24.840]The reason that this mutant performed so well
[00:06:27.520]could be due to electrostatic interaction.
[00:06:30.850]R is the amino acid arginine,
[00:06:33.130]and at a physiological pH of about 7.4,
[00:06:37.103]arginine's side chain is positively charged.
[00:06:40.590]At the same pH, the second carboxylic group of adipate
[00:06:44.620]is deprotonated and is negatively charged.
[00:06:48.200]So there is presumably an electrostatic interaction
[00:06:52.150]between these groups to improve substrate binding.
[00:06:56.260]The selection experiment, however,
[00:06:58.530]did lead to two false positives,
[00:07:01.740]T390G and G391R,
[00:07:05.920]which had no increased catalytic efficiency.
[00:07:10.650]In conclusion, these results show that the developed scheme
[00:07:14.550]is a successful strategy to identify CAR mutants
[00:07:17.390]with improved kinetic properties.
[00:07:19.650]In future work, this scheme could be applied
[00:07:22.030]to other substrates and other NADPH-oxidizing enzymes.
[00:07:26.910]And finally, I would like to acknowledge
[00:07:28.780]that this research would not have been possible
[00:07:31.170]without its UCARE award along with the grants
[00:07:34.150]from the National Science Foundation
[00:07:36.400]and the Nebraska Center for Energy Sciences Research.
[00:07:40.930]And I would like to thank my advisor, Dr. Niu,
[00:07:43.340]for being such a great mentor and resource.
[00:07:47.120]In addition, I would like to thank Levi Kramer,
[00:07:49.620]who was the grad student that has guided me
[00:07:52.260]throughout this project.
[00:07:53.800]He just graduated, so congratulations to him.
[00:07:57.700]And everyone in Niu's lab have been very helpful.
[00:08:00.930]I am very fortunate for being able to work with them.
[00:08:05.410]Thank you for listening and have a great rest of your day.

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Engineering Carboxylic Acid Reductase (CAR) via NADPH Recycling

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